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Oncotarget, December, Vol.2, No 12
Antitumoral activity of allosteric inhibitors of protein kinase
CK2
Virginie Moucadel 1 ' 23 *, Renaud Prudent 1 ' 23 *, Celine F. Saute! 123 , Florence
Teillet 123 , Caroline Barette 4 , Laurence Lafanechere 4 , Veronique Receveur-
Brechot 5 and Claude Cochet 1 ' 23
1 From INSERM, U1036, Biology of Cancer and Infection, Grenoble, F-38054, France
2 CEA, DSV/iRTSV, Biology of Cancer and Infection, Grenoble, F-38054, France
3 UJF-Grenoble 1, Biology of Cancer and Infection, Grenoble, F-38041, France
4 CEA, iRTSV/CMBA, Grenoble , F-38054, France
5 IMR Laboratory CNRS UPR3243, IMM, F-13402 Marseille cedex 20, France
Both authors contributed equally to this work
Correspondence to: Claude Cochet, email: claude.cochet@cea.fr
Keywords: Protein-kinase CK2, Inhibitors, Azonaphthalene, Cancer, SAXS
Received: November 24, 201 1 , Accepted: November 29, 201 1, Published: December 14, 201 1
Copyright: © Moucadel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT:
Introduction : Due to its physiological role into promoting cell survival and its
dysregulation in most cancer cells, protein kinase CK2 is a relevant physiopathological
target for development of chemical inhibitors. We report the discovery of
azonaphthalene derivatives, as a new family of highly specific CK2 inhibitors. First,
we demonstrated that CK2 inhibition (IC 50 = 0.4 pM) was highly specific, reversible
and non ATP-competitive. Small Angle X-ray Scattering experiments showed that
this inhibition was due to large conformational change of CK2a upon binding of
these inhibitors. We showed that several compounds of the family were cell-potent
CK2 inhibitors promoting cell cycle arrest of human glioblastoma U373 cells. Finally,
in vitro and in vivo assays showed that these compounds could decrease U373 cell
tumor mass by 83 % emphasizing their efficacy against these apoptosis-resistant
tumors. In contrast, Azonaphthalene derivatives inactive on CK2 activity showed no
effect in colony formation and tumor regression assays. These findings illustrate the
emergence of nonclassical CK2 inhibitors and provide exciting opportunities for the
development of novel allosteric CK2 inhibitors.
Background : CK2 is an emerging therapeutic target and ATP-competitive
inhibitors have been identified. CK2 is endowed with specific structural features
providing alternative strategies for inhibition.
Results : Azonaphthalene compounds are allosteric CK2 inhibitors showing
antitumor activity.
Conclusion : CK2 may be targeted allosterically.
Significance : These inhibitors provide a foundation for a new paradigm for
specific CK2 inhibition.
INTRODUCTION
Protein kinase CK2 plays critical roles in cell
growth and differentiation, apoptosis and oncogenic
transformation [1, 2]. Aberrant CK2 kinase expression
was associated with unfavorable prognostic markers in
prostate cancer [3] and in acute myeloid leukemia [4]
implicating CK2 in tumor formation and recurrence. Its
dysregulation in many other cancers together with its dual
function in promoting cell growth and in suppression of
apoptosis may be particularly relevant to its oncogenic
potential [5]. Further relevance for its role in cancer is
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indicated by its mediation of resistance to chemotherapy-
induced apoptosis [6]. Involvement of CK2 in other
diseases such as glomerulonephritis [7] or viral infections
(HIV, CMV, HPV, PV...) has been reported [8]. Based
on these observations, CK2 is now considered to be a
relevant physiopathological target amenable to therapeutic
intervention therefore supporting the identification and the
characterization of chemical inhibitors [9, 10]. Extensive
efforts have been focused on developing CK2 inhibitors
and several active, and in certain cases selective, ATP
site-directed compounds have been identified [11]. These
include condensed polyphenolic compounds such as
emodin and derivatives of hydroxycoumarins (3-carboxy-
4(lH)-quinolone),the indoloquinazoline derivative (IQA),
tetrabromocinnamic acid (TBCA), 4,5,6, 7-tetrabromo-l-
benzotriazole (TBB) and pyrazolo[l,5-a][l,3,5]triazine
derivatives [12]. Recently, our group and others reported
the identification of very efficient CK2 inhibitors with
good in vivo potency [13-15].
Beside ATP-competitive inhibitors binding to
the canonical ATP-site, small molecules targeting
different surfaces of kinases [16-18], including CK2
[19, 20] have been identified. Some of them bind to the
hydrophobic CK2B-binding cavity on CK2a, possibly
inducing an inactive conformation [21]. Indeed, an
inactive conformation of the catalytic CK2a subunit
was recently reported [22]. In this CK2a structure, it has
been suggested that the binding of small molecules to the
CK2B-docking site have an inhibitory impact on CK2a by
promoting its inactive conformation [21, 22]. Altogether,
these observations suggest the existence on CK2 of
different exosites distinct from the catalytic cavity that
can be targeted by small molecules to achieve functional
effects [19].
Using an automated screening, we have identified
azonaphthalene derivative compounds as new highly
potent CK2 inhibitors.
We report that azonaphthalene derivatives are
specific non ATP-competitive CK2 inhibitors. Small
Angle X-Ray Scattering analysis showed a major
conformational change of the kinase upon inhibitor
binding, Furthermore, several compounds of the family
are cell-permeable CK2 inhibitors promoting cell cycle
arrest of human glioblastoma U373 apoptosis-resistant
cells. Finally, we demonstrate that these compounds
decrease tumorigenesis in vitro and exhibit in vivo efficacy
in tumor growth assays.
These results show that a relevant allosteric
Recovery
MHmM)
1/[ATP] ((jM- 1 )
1/[Peptide] (mM' 1 )
Figure 1: Characterization of compound 1 as a reversible non-competitive CK2 inhibitor. A. Inhibition of the catalytic
activity of recombinant 36ng CK2a (♦) or 60ng CK2 holoenzyme (a 22 ) (■) by increasing concentrations of compound 1. B. Reversibility
of compound 1 inhibition. CK2a (2|ig) was incubated with 25uM compound 1 following by a gel-filtration chromatography. CK2 kinase
activity in input and flow-through were then assayed. C and D. Lineweaver-Burk inhibition plots of human recombinant CK2a by
compound 1. CK2 kinase activity was determined as described in the experimental section in the absence (♦) or in the presence of 2.5 (■), 5
( A ) and 7.5 (X) uM compound 1 with various concentration of ATP (C) or peptide substrate (D). The data represent means of experiments
run in triplicate with SEM never exceeding 10%.
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inhibition of CK2 activity can be achieved with non-ATP
competitive inhibitors expanding the options to modulate
this enzyme.
RESULTS
Identification of a new potent CK2 inhibitor
scaffold
The 2,860 compounds from the National Cancer
Institute Developmental Therapeutics Program small
molecule library were screened in an automated
luminescence-based in vitro kinase assay against the
human recombinant CK2 catalytic subunit CK2a as
previously published [21]. As a primary screen, CK2
kinase inhibitory activity was determined by measuring
the percentage of inhibition at a compound concentration
of 15 uM, using TBB and DMSO as positive and negative
controls respectively. A secondary screen performed at a
compound concentration of 1.5 |iM allowed the isolation
of 1 1 hits. Hit validation was performed at concentrations
of 1.5 uM using standard radiometric kinase assay
with high ATP concentrations (100 uM, K m for ATP of
recombinant CK2: 25 (0.M) to enhance the probability of
isolating non-ATP competitive compounds. This led to
the characterization of compound 1 as micromolar CK2
inhibitor (Figure SI). Compound 1 displays an IC 50 ~ 5
(0.M on CK2a and is equally potent on CK2a alone or
complexed with its regulatory CK2[3 subunit (Figure 1A).
Given that this active compound is hydrophobic,
planar and rigid with peripherical polar groups, we
investigated whether it could behave as promiscuous
inhibitors [23], acting via an aggregation mechanism [24,
25]. Since aggregate-forming inhibitors often display
steep dose-response curves and high Hill coefficients
[26, 27], we performed dose-response curves for CK2
inhibition. It was observed that compound 1 displayed
standard dose response curves with Hill slopes ranging
o
n
>
« 1
re
3
■a
tf> 0.5
<D
0£
51
101 151 201
Mutated residue
251
301
3
■2,
-=io- :
10"
10- ;
0.00
0.10
0.20 0.30
q (A" 1 )
0.40
CK2a
CK2a«1
Figure 2: Structure of CK2a and CK2a-l complex as determined using SAXS Ab initio shape restoration. A. Inhibitory
effect of compound 1 on WT and a panel of mutants with the following residues mutated to alanine (10, 24, 61, 71, 74, 76, 80, 123, 138,
160, 178, 195, 253, 268, 308, 311). Residual activity ratio is defined as the normalized activity of mutant CK2a divided by the normalized
activity of wild-type CK2 in presence of 5|xM compound 1. B. Experimental scattering spectrum of CK2a (black line, above) and of CK2a-
1 complex (black line, below), and theorical spectra of CK2a calculated by CRYSOL using the crystal structure of CK2a (PBD ID 1PJK)
as template (red line), and of the shape calculated by GASBOR for CK2a (blue line) and CK2a-l complex (green line). The curves have
been artificially shifted for better lisibility. C. Typical calculated shape obtained by GASBOR (green envelope) of CK2a (left panel) and
CK2a-l complex (right panel), superimposed with the atomic structures of CK2a (PDB ID 1PJK). The atomic structure is in red ribbon
representation.
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between 1.1 to 1.3 indicating that the compound likely
binds its target enzyme through a classical 1 : 1 inhibition
mechanism. Moreover size-exclusion chromatography
experiments indicated that CK2a inhibition by compound
1 is reversible (Figure IB), a feature that is rarely
associated with aggregating compounds. Taken together,
this rules out compound 1 as a promiscuous aggregator. To
define the mode of CK2 inhibition by compound 1, we
performed steady-state kinetic analysis. Lineweaver-Burk
inhibition plots showed that inhibition pattern of CK2a
by compound 1 was non competitive toward both ATP
and peptide substrate (Figure 1C and ID). Of note, at high
peptide substrate concentrations the plots were non-linear
suggesting that in the presence of saturating substrate
concentrations, the enzyme may be more susceptible to
A
DMSO TBB 1 1
48h 24h 24h 48h
inhibition by compound 1 .
SAXS analysis of CK2a-l complex
To determine the binding site of compound 1, we
tested its effect on a series of CK2a mutants (Figure
2A). This revealed that CK2a mutated on K78A, R80A
or L178A were more resistant to compound 1 inhibition,
suggesting that these residues located on helix aC and
on the activation segment are part of the compound 1
binding site. To get better insights into the molecular
interactions between CK2a and compound 1, we tried
to obtain X-ray crystallographic co-structure of CK2a-
1 complex. However, despite several crystallization
B
DMSO TBB 1 23
pSerl3-Cdc37
Cdc37
Tubulin
0 10 20 30 40 50 60 70 80 90 100
Compound Concentration (u.M)
100
■40
I 20
DMSO
0.5%
□ pGL5-GFP
□ pGL5-CK2a
TBB
50 uM
1
50 \lM
120
100
80
60
40
□ DMSO (0.5%) Dl^OuM)
0 TBB (50 □ 23(50^)
1 1*1
A549 H1299 LnCap MDA231 MCF-7
Cell Lines
Figure 3: Compound 1 is a cell-potent CK2 inhibitor and decreases cell viability in a CK2 dependent manner. A. HeLa
cells were plated and transfected with the CK2 activity reporter plasmid. One day after, medium was replaced with medium containing
increasing amounts of compounds and incubated for 24h or 48h. Then, cells were collected and the reporter phosphorylation status was
measured from whole cell extracts. Experiment was repeated 3 times. B. U373 cells were plated one day prior inhibitor addition. Twenty
four hours after compound addition, cells were collected and phospho-Cdc37, Cdc37 and tubulin levels were measured by immunoblotting.
Experiment was repeated twice. C. One day after plating, U373 cells were treated with increasing concentrations of TBB, 1, 23 or DMSO.
Two days after, living cells were counted as described. Results are given relative to the luminescence recorded for DMSO. Experiment was
done in triplicate and repeated twice. D. HeLa cells were transfected with a CK2rx or a GFP-expressing plasmid. One day later, medium
was replaced with medium containing 0.5% DMSO or 50 uM TBB, 1. Two days after, living cells were counted as in C. Results are given
relative to the luminescence recorded for DMSO in each condition. Experiment was done in triplicate and repeated twice. E. One day after
plating, cells were treated with 50 uM TBB, 1, 23 or 0.5% DMSO. Two days after, living cells were counted as in A. Results are given
relative to the luminescence recorded for DMSO in each cell lines. Experiment was done in triplicate and repeated twice.
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screening campaigns, we were unable to get diffracting
crystal containing CK2a complexed with compound 1 .
Therefore, we performed Small Angle X-Ray
Scattering (SAXS) experiments on rhCK2a AC (l-335)
and rhCK2a AC (l-335)-l complex (Figure 2B). SAXS
is indeed a very appropriate tool to assess the structural
properties (dimensions, flexibility and overall shape) of
proteins in solution. The Guinier law allows the scattered
intensities to be approximated, at low scattering angles
to determine the radius of gyration (R G ). When applied
to CK2a, R values calculated for different protein
concentrations revealed that upon compound 1 binding,
R Q (extrapolated to zero concentration) increases from
32.8±1.2 A to 35.6±1.1 A at 20°C and the corresponding
Maximal diameter (Dmax) increases from68±3 Ato96±4
A. As probed by kinase assay, compound 1 inhibition
is reversible and Kratky plot of CK2a-l SAXS data
exhibits a bell-shaped profile typical of globular protein.
Thus, a denaturation of CK2a by compound 1 can be
ruled out. The overall shape of rhCK2a AC (l-335) and
rhCK2a AC (l-335)-l complex were calculated ab initio
using the program GASBOR (Figure 2C). Different
runs gave similar shapes. Averaged calculated shape of
rhCK2a AC (l-335)-l complex superimposed with the
X-ray structure of rhCK2a AC (l-335) (PDB ID 1PJK)
shows that CK2a undergoes a conformational change,
leading to a distorted shape. In this conformation, CK2a
could be inactive due to non-optimal spatial arrangement
Table 1: Features that confer inhibitory potency to compound 1. A. CK2 kinase assays were performed in the absence or
presence of 10 uM compound. Results are expressed as a percentage of the control activity without inhibitor. B. Compounds that are cell
permeable CK2 inhibitors. CK2 inhibition was monitored as in
A
B
Compound
4 |
7
8
1 1
4
CK2 activity (%)
1
N=N
OH
H
SO3H
H
N0 2
H
H
N=N
OH
H
H
80
N=N
OH
H
SO3H
H
H
H
H
N=N
OH
H
H
18.3
N=N
OH
H
SO3H
H
H
H
H
N=N
OH
H
H
3.9
1 4
N=N
OH
H
SO3H
H
N0 2
H
H
OH
N=N
H
H
0.2
N=N
OH
H
SO3H
H
H
H
H
OH
N=N
H
H
14.2
N=N
OH
H
SO3H
N0 2
H
H
H
OH
N=N
H
H
1.3
7
NH 2
OH
H
SO3H
H
H
H
H
none
83
8
N=N
OH
H
H
H
H
H
H
N=N
H
H
H
68.5
N=N
OH
H
SO3H
H
H
H
H
A
15.8
10
N=N
OH
H
SO3H
H
H
H
H
B
48.7
11
N=N
OH
H
SO3H
H
H
H
H
OH
N=N
COOH
H
1.2
12
N=N
OH
S0 3 H
H
H
SO3H
H
H
N=N
H
H
H
28.5
13
N=N
OH
H
H
H
SO3H
H
SO3H
N=N
H
H
H
58.3
14
H
N=N
H
H
H
SO3H
H
H
N=N
OH
H
H
20.2
15
N=N
H
H
H
H
H
H
SO3H
N=N
OH
H
H
14.1
16
N=N
H
H
H
H
H
SO3H
H
N=N
OH
H
H
12
17
N=N
H
H
H
H
SO3H
H
H
N=N
OH
H
H
7.4
18
N=N
H
H
H
SO3H
H
H
H
N=N
OH
H
H
4.5
19
N=N
OH
H
H
H
SO3H
H
H
N=N
H
H
H
0.8
20
N=N
OH
SO3H
H
H
SO3H
H
H
N=N
H
H
SO3H
7.6
21
N=N
OH
H
H
H
SO3H
H
SO3H
N=N
H
H
SO3H
25.7
22
OH
N=N
H
H
SO3H
H
H
H
N=N
H
H
SO3H
20.9
23
N=N
H
H
NH 2
H
SO3H
H
H
C
10
A : p-cresol; B : 3-methyl-l-phenyl-lH-pyrazol-5-ol ; C : 4-phenylphenol
B
uM
24h
48 h
50
100
200
50
100
200
1
+
+
+
+
+
+
+
4
+
+
+
+
+
+
+
ND
ND
ND
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of its catalytic site. Alternatively, some domain movement
may be impaired impeding catalysis.
Effect of compound 1 on cellular CK2 kinase
activity
To evaluate the efficacy of compound 1 to target CK2
into living cells, we used a cellular CK2 activity assay
[28]. Compound 1 tested at increasing concentrations for
24 or 48 h was active on cellular CK2 activity (Figure
3A). This was also confirmed by immunoblotting
using a phosphospecific antibody recognizing Cdc37
phosphorylated on Serl3 which is specifically targeted
by CK2 [29]. Thus, Serl3-Cdc37 phosphorylation
status can be used as a surrogate cellular CK2 activity
assay [29]. We found that under similar conditions (50
|j,M, 48h incubation), compounds 1 like TBB, reduced
drastically Cdc37 phosphorylation on Serl3. Compound
23, an analogue of compound 1 which is known to be cell-
permeable [30] was inactive both on recombinant CK2a
and on cellular CK2 activity, (Figure 3B).
Compound 1 decreases cell viability in a CK2-
dependent manner
CK2 inhibitors are known to decrease cell viability.
Thus, we measured HeLa cell viability after 48h treatment
with increasing amount of TBB or compound 1 . Compound
23 was used as negative control. Viability of HeLa cells
exposed to compound 1 or TBB showed similar decreased
viability (IC 50 3 3 |iM and 36 (xM respectively) (Figure
3C). To determine whether the reduction of cell viability
triggered by compound 1 was mediated by CK2 inhibition,
we transfected HeLa cells with a CK2a or a GFP-
expressing plasmid. Cells expressing exogenous CK2a
displayed a better survival when treated with compound
1 than control cells (Figure 3D). Then, we measured the
effect of compound 1 on the viability of different known
tumor cell lines (Figure 3E). At a concentration of 50 uM,
compound 1 decreased viability of most tested cell lines
and was even more efficient than TBB, especially in p53
mutant cell lines like U373 and to a lesser extent HI 299
and MDA231. This decreased viability was correlated
with a down regulation of CK2 activity attested by Cdc37
Serl3 phosphorylation which was drastically reduced
in U373 cells exposed to compound 1 (Figure 3B). In
contrast, this reduction of Cdc37 Serl3 phosphorylation
was not observed with 50 (.iM TBB or compound 23.
Structure Activity Relationship of azonaphthalene
compounds on CK2a
To define the features that confer inhibitory potency
to compound 1, we tested several derivatives in vitro and
in the context of a complex cellular milieu (Table 1 and
Table SI). First, compounds showing a CK2 inhibiting
activity in vitro were tested at increasing concentrations
for 24 or 48 h in the cellular CK2 assay. Five compounds
inhibited CK2 in this assay: 1, 3, 4, 5 and 6 (Figure 3A
and 3B, Table IB). The most potent compounds were: 1
> 4, 5 >3 > 6.
Given that this class of compound possess a
symetrical scaffold made of two naphthyl parts (A and
B) bound by an azo function, we first investigated the
requirement of part A. Compound 6, which possesses a
nitro function at position A5 was fully active both in vitro
and in the CK2 cellular assay. Moreover, compound 2
which lacks nitro function at position A6 remains active
in vitro and compound 3 (which is zinc complex of
compound 2) remains active in our cellular assay. Since
nitro function is not necessary, it will be possible to remove
it thereby avoiding possible carcinogenic metabolite.
To gain insight into the requirements of the hydroxyl
function in position A2 and the sulfonic acid in position
A4, several derivatives of compound 1 were tested.
Compound 8 which lacks the nitro, hydroxyl and sulfonic
acid functions was notably inactive. This indicates a clear
requirement for hydroxyl and/or sulfonic acid functions.
Position variations of sulfonic acid function (compounds
14-18) lead to in vitro active compounds, indicating a
requirement for sulfonic acid function. However none
of these compounds are active in the CK2 cellular assay.
Thus, hydroxyl function at position A2 and sulfonic acid
at position A4 is, up to now, the optimal combination to
get cell-potent compounds. Moreover, both compounds 3
and 5 are active both in vitro and in a CK2 cellular assay,
indicating some tolerance in the position of the azo linker.
We next investigated the requirements of part B.
Removal of this moiety is detrimental (compound 7). This
indicates that this part of the molecule possesses functions
conferring activity both in vitro and in our CK2 cellular
assay. Testing of compound 1 1 reveals that an additional
carboxylic function enhances the in vitro activity.
Compounds 9 and compound 10 were much less active.
This suggests that replacement of the naphthalene core
is also detrimental. Moreover compounds 9-11 are also
inactive in the CK2 cellular assay showing that Naphth-
2-ol is, so far, the sole substituent conferring activity in a
cellular context.
Collectively, it appeared from this SAR analysis
that compound 4 was the most potent inhibitor in this
compound series, being 10-fold more powerful than
compound 1. Therefore, further studies were focused on
compound 4.
Analysis of CK2 inhibition by compound 4
Dose-response curves for CK2 inhibition showed
that compound 4 displayed an IC 50 ~0.4 uM on CK2a
(Figure S2). To define the mode of CK2 inhibition by
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compound 4, we performed steady-state kinetic analysis.
It appeared that inhibition of CK2a by compound 4 was
not competitive toward both ATP and peptide substrate
(Figure 4). Again, plots were non linear with regards to
peptide substrate concentrations. CK2 inhibition was
strongly increased at high peptide substrate concentrations
(Figure S3), a feature in line with the mode of inhibition
of compound 1 (Figure ID and S3).
Selectivity profiling of compound 4
Specificity is a major concern for kinase inhibitor
development. The kinase panel tested included members
of major human protein kinase families [31] (Fig S4). This
screening revealed that at 5uM, compound 4 inhibited
CK2 by more than 95%, but had almost no effect on
the other protein kinases tested (Table 2). Such high
selectivity rules out compound 4 as promiscuous inhibitor.
We calculated a previously described metric for kinase
inhibitor selectivity based on the Gini coefficient [32].
The Gini score reflects, on a scale of 0 to 1 , the degree to
which the inhibitory activity of a compound (calculated
as the sum of inhibition of all kinases) is directed toward
only a single kinase (a Gini score of 1) or is distributed
equally across all tested kinases (a Gini score of 0). Not
surprisingly, the calculated Gini coefficient for compound
4 was 0.803 (Fig S5), a value among the highest for CK2
inhibitors [33, 34] and for kinase inhibitors in general [35].
Such conclusion is also supported by the "hit rates" i.e.
the number of kinases inhibited by >50% by compound 4
using the 42 kinase panel: its value is 1, highlighting high
specificity and suggesting that this compound might have
characteristics of a uni-specific kinase inhibitor [35].
Analysis of compound 4 binding site
Despite several attempts we could not get diffracting
crystals to solve the 3D structure of a CK2-4 complex.
Therefore, to delineate a potential binding site of compound
4, we tested its inhibition on a series of CK2a mutants
(Figure 5). Strikingly, this revealed that CK2 mutants that
were found insensitive to compound 1, were also resistant
to compound 4 inhibition. This strongly predicts that both
compounds have a common binding site located on helix
aC and on the activation segment of CK2a.
Taken together this data indicates that compound 4
and compound 1 have a common, or at least overlapping
binding site and inhibit CK2 in a similar manner.
Compound 1 and 4 promotes cell cycle arrest
We then focus our attention on the U373 cell line
because they were particularly responsive to compound
1. These cells originate from glioblastoma, an aggressive
tumor of the brain with poor survival prognostic. They
harbor a p53 mutant isoform and are particularly resistant
to drug-induced apoptosis [36]. First, we explored
whether compound 1 could inhibit their cell cycle as it was
already observed for compound 4 in endothelial cells [37]
and A431 epidermoid carcinoma cells [38]. Cells were
1/[ATP] (nM !) l/[Peptide] (nM 1 )
Figure 4: Characterization of compound 4 as a reversible non-competitive CK2 inhibitor. Lineweaver-Burk inhibition
plots of human recombinant CK2a by compound 4. CK2 kinase activity was determined as described in the experimental section in the
absence (♦) or in the presence of 0.2 (■), 0.4 ( ▲ ) and 0.8 (X) \iM compound 4 with various concentration of ATP (A) or peptide substrate
(B). The data represent means of experiments run in triplicate with SEM never exceeding 10%.
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treated with 50 (0.M of compound 1 for 48h or equivalent
concentration of TBB, compounds 23, 4 or DMSO and
then labeled with propidium iodide. U373 cells exposed
to compounds 1 or 4 exhibited a cell cycle arrest with a
substantial accumulation in mid-S-phase (Figure 6A).
Azonaphthalene derivatives inhibit colony
formation and tumor growth
To assert the potential clinical interest of
azonaphtalene derivatives, we first tested their ability to
inhibit colony formation using soft agar assay as an in
vitro surrogate of tumorigenesis. U373 cells can grow
without anchorage and form colonies in soft agar culture
reflecting their malignant properties. Colony formation
assays were performed using compounds 1, 4, and
compound 23 and TBB as controls. Both Compound 1 and
4 inhibited colony formation in a dose dependent manner
while TBB or compound 23 were without effect at the
same concentration (Figure 6B).
The NCI DTP website shows that compounds 1 and
4 that were the most active in a colony formation assay,
have a good pharmacological profile ( http://dtp.nci.nih.
gov/docs/invivo/invivoscreen.html ) a property which was
confirmed by the group of Atassi [38]. Compounds 3, 5
and 6 were not evaluated by the NCI.
We next tested the azonaphthalene compounds
in a murine U373 tumor regression assay. We chose
compound 4 for further in vivo investigations because
of its high potency and specificity. The data illustrated in
Figure 6C show that intratumoral injection of compound
4 could decrease tumor incidence across all time points
compared to mice injected with PBS. After 3 weeks, mice
injected with compound 4 had tumors that were six times
smaller than those injected with PBS (Figure 6C). Western
blot analysis showed that Cdc37 Serl3 phosphorylation in
U373 tumors, was significantly reduced in mice injected
with compound 4 compared to mice injected with PBS
(Figure 6C).
To assess whether the anti-tumoral effect was
related to CK2 inhibition, we wanted to inject mice
with compound 23 that was used as inactive control in
colony formation assay and CK2 activity cellular assay.
However, a previous study [30] has shown that this
compound displays antitumoral effect via inhibition
of neoangiogenesis. Therefore, mice were treated with
compound 9 (Calmagite) which was also inactive in the
CK2 activity cellular assay. No significant anti-tumoral
Table 2: Kinase Selectivity profile of compound 4. Residual kinase activity determined in the presence of 5 uM inhibitor is
expressed as percentage of the control activity without inhibitor. Final concentration of ATP in the experiment was 100 |iM.
Protein kinase
Residual
activity {%)
Protein kinase
Residual
activity (%)
Protein kinase
Residual
activity (%)
Protein kinase
Residual
activity (%)
CK2a2(h)
4
PKBa(h)
95
PDGFRa(h)
106
LOK(h)
114
PKA(h)
57
MKK7B(h)
96
ROCK-I(h)
106
CDK6/cyclinD3(h)
115
Lyn(h)
63
TAKl(h)
96
CDKl/cyclinB(h)
109
CHKl(h)
121
AMPKal(h)
65
c-RAF(h)
97
MEKl(h)
110
p70S6K(h)
122
CKlyl(h)
82
Pim-l(h)
97
PAK2(h)
110
ASKl(h)
124
MSTl(h)
88
eEF-2K(h)
98
CaMKI(h)
111
PKC6(h)
128
NEKll(h)
89
EGFR(h)
98
mTOR(h)
111
CDK7/cyclinH/MATl(h)
129
Plk3(h)
90
PKCa(h)
100
MKK6(h)
112
IRAK4(h)
145
DRAKl(h)
94
CDK2/cyclinA(h)
101
MLKl(h)
112
ALK(h)
148
JAK2(h)
94
EphA5(h)
101
KDR(h)
113
Fyn(h)
95
Abl(h)
103
IKKct(h)
114
1.5
O
+J
TO
i-
>■
>
u
■a
« 0.5
0£
♦
51
101 151 201
Mutated residue
251
301
Figure 5: Compound 4 shares overlapping binding
site with compound 1. Inhibitory effect of compound 4
on WT and a panel of mutants with the following residues
mutated to alanine (10, 24, 61, 71, 74, 76, 80, 123, 138,
160, 178, 195, 253, 268, 308, 311). Residual activity ratio
is defined as the normalized activity of mutant CK2a
divided by the normalized activity of wild-type CK2a
in presence of 0.4|xM compound 4. Normalized activity
represents the percentage of residual activity in presence
of 0.4 |iM compound 4.
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Oncotarget 2011; 2: 997 - 1010
effect could be observed between mice treated with
compound 9 and control vehicle (Figure S6). Collectively,
these observations suggest that tumor growth inhibition
by compound 4 is likely related to CK2 inhibition.
DISCUSSION
Abnormally high CK2 expression provides
cancer cells with an environment favorable for tumor
development [39]. Therefore development of CK2
inhibitors may represent an interesting approach for
the treatment of cancer. Most CK2 inhibitors are ATP-
competitive but new development in the kinase field has
raised interest in exploiting alternative mode of inhibition
[40]. In this work, we describe a family of chemicals that
are non-ATP competitive and cell potent CK2 inhibitors
with good profile for their in vivo use.
The exquisite selectivity of compound 4 revealed by
its high Gini coefficient, could reflect unique features of
CK2 and be related to the allosteric mechanism of action of
this inhibitor. Although strong kinase selectivity may not
be essential for efficacy of therapeutic agents, it is critical
for tool compounds used to elucidate kinase biology.
Site-specific mutations of Lys74Ala, Arg80Ala
or Leul78A in CK2a showed that these mutant forms
exhibit some resistance toward compound 1 and 4
inhibition. Interestingly, these three mutations are
located in or close to structural elements that are crucial
for CK2 activity, namely the substrate binding site, the
RD and DWG motifs [41, 42]. By interfering with these
regulatory elements, compound 1 and 4 may stabilize a
non-productive catalytic form of CK2a.
We consistently observed parabolic inhibition plots in
the presence of increasing peptide substrate concentrations,
a feature that may arise from allosteric effects. In this
Proportion of cells (%) in
Sub-Gl
Gl
S
G2/M
DMSO
1.1+0.09
65.9±0.7
7.5±1.1
25.2±L9
TBB
2.1±0.02
70.6±3.8
3.2+0.3
22.3±2.9
1
4.7±1.3
61.2±0.7
16±1.1
17.4±1.4
4
4.3±0.9
56.5±0.9
19.6±1.2
14.5±2.3
23
0.9±0.13
75.3±0.5
4.3±1
22.3+2.1
100 |
0)
'c
80
o
o
u
60
o
01
J2
40
E
z
20
>
0
01
cc
0.5% 10|iM 25|iM 50|iM 50|iM 50|iM 50|aM
DMSO 1
TBB
23
III III
4 or PBS
Injections
55 6b
Days
post -first injection
Figure 6: Azonaphthalene derivatives promote cell cycle arrest, reduce colony formation and exert anti-tumoral effect
in vivo. Nearly confluent U373 cells were treated with compounds or equivalent amount of DMSO. After 48h, cells were harvested and
labeled with propidium iodide. DNA content was analysed with FACScalibur and Cell Quest software. Experiment was repeated three
times. Proportion of U373 cells in sub-Gl, Gl, S and G2/M phases are summarized. B. Colony formation assay by soft agar culture. U373
cells were poured on an agarose layer (0.6%) mixed with agarose (0.3%) containing compounds for colony formation assay (soft agar).
Fifteen days later, colonies (more than 20 cells) were counted in 10 fields/well. Experiment was performed in duplicate at least three times.
C. Athymic nude mice were inoculated subcutaneously into the right flank with 7.5xl0 5 U373 cells. When tumor reaches ± 50 mm 3 , animals
were treated intratumorously 3 times weekly for 2 weeks, with compound 4 dissolved in PBS ( 1 mg/20 |il/injection) or PBS (control group).
Tumor volume was determined twice weekly. Results represent the average of the tumor volume of 5 mice per group. Insert: representative
western blots. Tumor extracts were analyzed by western blot for expression of tubulin, Cdc37 and P-Serl3-cdc37 [29].
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1005
Oncotarget 2011; 2: 997 - 1010
scenario, a substrate-dependent "conformation selection"
would favor inhibitor binding.
Our SAXS analysis showed that the overall shape
of a CK2a-compound 1 complex is different from active
CK2a shape obtained either by SAXS (this work) or
by X-ray crystallography [43]. Moreover, the shape of
the CK2a-compound 1 complex is also different from a
structure of an inactive CK2a conformation [22]. Taken
together, the binding of compound 1 may distort critical
regulatory elements and lock the kinase in a new non-
productive conformation. Further structural analysis will
be required to precisely reveal the binding site of these
inhibitors.
Azonaphthalene derivatives have been described
as nonpeptidyl thrombopoietin mimics [44]. We show
that molecules belonging to this family of chemicals are
cell-permeable and can target the cellular CK2 activity.
A panel of tumor cell lines exposed to compound 1
showed a decreased viability that was correlated with a
down regulation of CK2 activity. Indeed, involvement of
CK2 in cell cycle regulation is well documented. CK2 is
required at multiple transitions in the cell cycle (including
G0/G1, Gl/S and G2/M). We found that U373 cells
exposed to compounds 1 or 4 exhibited a cell cycle arrest
with a substantial accumulation in mid-S-phase. These
observations are in accordance with similar effects of
compound 4 previously described in endothelial cells [37].
Whether activation of apoptosis that occurs in HeLa cells
treated with high doses of compound 1, is a consequence
of a cell cycle arrest or direct promotion of apoptosis
remains to be established. Of note, the viability of U373
cells which are particularly resistant to drug-induced
apoptosis [36] was strongly affected by compound 1 and
this effect was correlated with a down regulation of CK2
activity.
Compound 1 inhibited U373 cell colony formation in
a dose dependent manner. Of note, CK2 phosphorylation of
HSP90 modulates chaperone function and drug sensitivity
[45]. By promoting activities of several oncokinases,
the Cdc37/Hsp90 chaperone complex contributes to the
acceleration of cell proliferation observed in cancer cells.
Like CK2, Cdc37 is over-expressed in cancer cells and a
target for cancer therapy [46]. To interact properly with
client kinases, Cdc37 must be phosphorylated on Serl3
by CK2. We showed that compound 1 decreases Cdc37
phosphorylation on Serl3 both in U373 cells and in
tumor-bearing mice. We can hypothesize that at least part
of the anti tumorigenic effect of this class of compound
could involve inhibition of Cdc37 phosphorylation.
Interestingly, it was recently reported that Sarcoma 180
tumors were sensitive to compound 1 showing a -50%
tumor regression in treated mice (NCI DTP website).
Cdc37 Serl3 phosphorylation in glioblastoma tumors was
also significantly reduced in mice injected with compound
4 suggesting that at least part of the antitumoral effect of
this class of inhibitors could involve inhibition of Cdc37.
We cannot exclude that the effect of azonaphthalene
derivatives could be also explained by the partial inhibition
of other targets. However, to date compound 4 is among
the most selective CK2 inhibitors published.
In this work, we showed that compound 4 induced a
significant inhibition of glioblastoma tumor growth that was
correlated with CK2 inhibition. Of note, compound 4 was
reported to inhibit angiogenesis in a chick chorioallantoic
membrane assay and proliferation of primary endothelial
cells, A431, L1210 and M5076 cancer cells [38]. In this
study, the authors suggested that the effect of compound
4 could involve cell cycle perturbation through inhibition
of topoisomerase II catalytic activity [38]. Interestingly,
topoisomerase II is a known CK2 target [47].
In conclusion, we described the discovery of a
family of Azonaphthalene derivatives that perturb CK2a
conformation thereby blocking productive binding
of substrates. These compounds may be useful as
research tools in probing CK2 conformational plasticity
because our kinetic analysis showed a mechanism of
action consistent with an allosteric mode of inhibition,
highlighting the opportunity of exploiting different CK2
inhibition mechanisms [19]. Although further structural
analysis will be required to reveal the binding site of these
inhibitors, their therapeutic potential is emphasized by
their efficacy in growth inhibition of apoptosis-resistant
tumors. Further optimization of these compounds and the
results from further in vivo testing may aid in the design
of other classes of CK2 inhibitors and should reveal their
efficacy in additional specific models.
METHODS
Cell culture
HeLa (cervical adenocarcinoma), U373
(glioblastoma) and MDA23 1 (breast adenocarcinoma) cell
lines were cultivated in Dulbecco's medium (Invitrogen)
Life Technologies, Inc.) while A549 (lung carcinoma),
HI 299 (lung carcinoma) and LnCap (prostate carcinoma)
cell lines were cultivated in RPMI (Invitrogen) Life
Technologies, Inc.). Each medium was supplemented with
10% (v/v) fetal calf serum (FBS, Bio West). MCF7 (breast
adenocarcinoma) cell line was cultivated in Dulbecco's
medium with insulin (10|ig/ml).
High-throughput screening
High-throughput screening of the NCI Diversity
Set chemical library (2,860 compounds) was performed
as previously described [48]. Active fractions from the
primary screen were retested from freshly made solutions.
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Cellular CK2 activity assay
CK2a production and purification
A cellular CK2 activity assay was performed as
previously described [28].
Western Blot
Western-blotting was performed as previously
described [11] using the following antibodies: anti-CK2a
antibody [3] at 1/5000, oranti-Serl3-phospho Cdc37 [29]
at 1/3000, anti-Cdc37 (Santa Cruz, scl3129) at 1/2000
and anti-tubulin (YL1/2, Abeam) at 1/50000.
Kinase selectivity profiling
Kinase selectivity of compound 4 was assessed using
the Kinase Profiler service offered by Millipore which
utilizes a radiometric filter-binding assay. The assays
were performed at 100 uM ATP in the presence of 5 uM
inhibitor. Inhibition, expressed as the percent of activity
determined in the absence of inhibitor, was calculated
from the residual activity measured in the presence of 5
uM inhibitor.
Selectivity parameters
Lorenz curves were derived from the selectivity
data. Gini coefficients and Hit rates were calculated as
described in [32].
Cell Viability
Cell viability assay was performed as described
previously [28]
Cell cycle distribution analysis
Cell cycle distribution analysis was performed as
described previously [15].
Soft agar assay
Soft agar assay was performed as described
previously [15].
CK2 Phosphorylation assays
CK2 radiometric kinase assay based on conventional
filter-binding assay was performed as previously described
[20].
GST-rhCK2a AC (l-335) expression in E.Coli BL21
cells after induction with IPTG and lysis were performed
as described in [15]. Supernatant was added onto
glutathion-sepharose beads (Amersham). After overnight
incubation, beads were washed by 40mM Tris pH 7.5,
150 mM NaCl and cleavage reaction was performed
on column (1 unit of thrombin.mL A of beads (Sigma,
T6634) in PBS containing 200 mM NaCl, 2% glycerol
and 1 mM DTT for 4h at 4°C). Following elution (50 mM
Tris pH 7.5, 150 mM NaCl) the proteins were loaded on
a heparine-sepharose column equilibrated in the elution
buffer. Proteins were then eluted with a linear gradient of
0. 15-1M NaCl in 50 mM Tris pH 7.5. The active fractions
were pooled and concentrated with a Centricon Plus-70mL
(Millipore). Production and purification of GST-rhCK2a
mutants were carried out using the same protocol.
SAXS experiments
SAXS experiments were performed at the SOLEIL
Synchrotron (Saclay, France) on beamline SWING. The
wavelength X was 1.0 A (1 A=0.1 nm) and the sample-to-
detector distance was 1924 mm giving access to scattering
vectors q ranging from 0.009 to 0.47 A-l.The scattering
vector is defined as q = 4n IX sin8, where 28 is the
scattering angle. The detector was an AVIEX170170 CCD
detector, and 40 successive frames of 4.2 ms exposure
were recorded for each sample. The samples were injected
with an automatic sample-changer and circulated through
an evacuated quartz capillary between each frame to avoid
radiation damage.
Protein concentration of CK2a (in 25 mM Tris pH
8.5, 0.2 M NaCl, 1 mM DTT and 10% glycerol) was
varied from 1.25 to 10 nig-mF 1 in order to check for
interparticle interactions. The protein concentration of
CK2a in the presence of 500 uM compound 1 (CK2a/l
ratio 1:2) was varied from 1.25 to 10 mg mr 1 . The radius
of gyration R Q was derived by the Guinier approximation
I(q)=I(0yexp(-q 2 R G 2 /3) for qR a <l.0. The distance
distribution function P(f) was calculated by the Fourier
inversion of the scattering intensity I(q) using GNOM
[491. The maximum diameter of the macromolecule, D ,
L J 3 max 3
was also determined [50].
Using the program GASBOR [51], the overall shapes
of CK2a alone or in complex with compound 1 were
restored from the experimental data. Ten independent fits
were run with no symmetry restriction, and the stability of
the solution was checked. The atomic structure of CK2a
(PDB ID 1PJK) was then fitted in the averaged calculated
shape, using SUPCOMB [52]. The theoretical scattering
curve of CK2a (PDB ID 1PJK) was calculated using
CRYSOL [53].
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Murine xenograft tumor growth assays
All experimental procedures adhered to our local
ethical committee (Comite regional d'efhique pour
l'Experimentation animale CREEA, Rhone Alpes -
protocol n°315). Female Harlan athymic nude mice (6-8
weeks) were inoculated subcutaneously into the right
flank with 7.5xl0 5 U373 cells. When tumors reaches ± 50
mm 3 (volume = length x width x height), animals were
treated intratumorously 3 times weekly for 2 weeks, with
compound 4 dissolved in PBS (1 mg/20 Lil/injection) or
PBS (control group). Body weight and tumor volume
were determined twice weekly. The experiment was
terminated when tumor volume was about 1000 mm 3 .
Data are expressed as means and s.d. and were analysed
with Student's ?-test; significance is defined as /?<0.05.
Tumor extracts were prepared in RIPA buffer and
analyzed by western blotting.
ABBREVIATIONS
CK2: Casein Kinase 2; SAXS: Small Angle X-ray
Scattering ; TBB : 4,5,6,7-TetraBromo-lH-Benzotriazole
; R Q : radius of gyration.
ACKNOWLEDGMENTS
The authors thank Dr. V. Colin for her help during
FACS experiment and H. Pointu and his staff for animal
health care. Thanks also to the ChemAxon company ( http://
www.chemaxon.com ) which has allowed the academic
Tamis software team to freely use the MarvinView
package, We acknowledge SOLEIL for access to the
Beamline Swing and Javier Perez for assistance. The
authors gratefully thank the National Cancer Institute
Developmental Therapeutics Program for providing
chemical libraries.
GRANT SUPPORT
This work was supported by the Institut National
de la Sante et de la Recherche Medicale (INSERM), the
Centre National pour la Recherche Scientifique (CNRS),
the Commissariat a l'Energie atomique (CEA), the Institut
Curie, the Ligue Nationale Contre le Cancer (equipe
labellisee 2010), the Institut National du Cancer (grant
number 57). RP gratefully acknowledges support from the
Fondation pour la Recherche Medicale Fellowship.
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